Methods for making mixed allergen compositions

ABSTRACT

Methods of making mixed allergen compositions, e.g., substantially aerobic organism free mixed allergen compositions, and the resulting allergen composition, are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of the filing dates of U.S.Provisional Application No. 62/533,826, filed Jul. 18, 2017, and U.S.Provisional Application No. 62/551,395, filed Aug. 29, 2017. The entirecontents of each of these applications are hereby incorporated byreference herein in their entireties.

BACKGROUND

Allergy is a disorder of the immune system characterized by theoccurrence of allergic reactions to normally harmless environmentalsubstances. Allergies are caused by allergens, which may be present in awide variety of sources, including but not limited to pollens or otherplant components, dust, molds or fungi, foods, additives, latex,transfusion reactions, animal or bird danders, insect venoms,radiocontrast medium, medications or chemicals. Common allergicreactions include eczema, hives, hay fever, asthma, and reactions tovenoms. Mild allergies like hay fever are highly prevalent in the humanpopulation and cause symptoms such as allergic conjunctivitis,itchiness, and runny nose. In some people, severe allergies toenvironmental or dietary allergens or to medication may result inlife-threatening anaphylactic reactions and potentially death, if leftuntreated.

A food allergy is an adverse immune response to a food allergen, e.g., afood protein. Common food allergens are found in shellfish, peanuts,tree nuts, fish, milk, eggs, soy and fresh fruits such as strawberries,mangoes, bananas, and apples. Immunoglobulin E (IgE)-mediated foodallergies are classified as type-I immediate hypersensitivity reactions.These allergic reactions have an acute onset (from seconds to one hour)and the accompanying symptoms may include angioedema (soft tissueswelling of the eyelids, face, lips, tongue, larynx and trachea), hives,itching of the mouth, throat, eyes, or skin, gastrointestinal symptomssuch as nausea, vomiting, diarrhea, stomach cramps, or abdominal pain,rhinorrhea or nasal congestion, wheezing, shortness of breath, ordifficulty swallowing, and even anaphylaxis, a severe, whole-bodyallergic reaction that can result in death. It is estimated that 1 outof 12 children under 21 years of age have a doctor's diagnosis of foodallergies, and over $24 billion is spent per year on health care costsfor food allergic reactions, largely due to about 90,000 visits to theER per year in the U.S. due to food induced anaphylactic symptoms.Moreover, there are still deaths that occur every year due to fatal foodallergies.

Accordingly, there exists a need in the art for allergen compositionsthat can prevent and/or treat allergies, and methods for making allergencompositions to prevent and/or treat allergies

SUMMARY

The disclosure is directed, at least in part, to a method for producinga substantially aerobic organism free mixed allergen composition. Forexample, this disclosure provides a method for producing a substantiallyaerobic organism free mixed allergen composition comprising: providing amixed allergen composition comprising 6 or more allergens, e.g., 6 to 20allergens, and a bulking agent, wherein the mixed allergen compositioncomprises at least 6% fat content; milling the mixed allergencomposition in a conical mill to obtain a milled composition withsubstantially consistent particle size; and applying microwaves or radiofrequency interference to the milled composition so that the milledcomposition is heated to at least 190° F. for at least 30 minutes,thereby to obtain the substantially aerobic organism free mixed allergencomposition. In certain embodiments, the mixed allergen compositioncomprises at least 12% fat content.

As part of a contemplated method, milling the mixed allergen compositionmay comprise using a rotor speed of about 9000 RPM and/or passing themixed allergen composition through a screen with an opening size ofabout 0.033 inches. As part of a contemplated method, milling the mixedallergen composition may comprise applying a vacuum suction through theconical mill.

A contemplated method may further comprise wetting the milledcomposition, e.g., to a moisture content of about 6% to about 9%, e.g.,about 9%, and/or a water activity of about 0.65 to about 0.8, e.g. about0.75 to about 0.8, prior to applying microwaves or radio frequencyinterference to the milled mixed allergen composition. A contemplatedmethod may further comprise pressurizing the milled composition, e.g.,to at least 30 psi for at least 30 minutes. In certain embodiments,applying microwaves or radio frequency interference to the milledcomposition and pressurizing the milled composition are conductedsimultaneously.

As part of a contemplated method, the milled composition may be heatedto at least 200° F. for at least 60 minutes, e.g., to at least 230° F.for at least 60 minutes. As part of a contemplated method, the milledcomposition may be heated to no more than 250° F. and/or for no morethan 2, 5, 10, 30, 60, 120 or 1000 minutes. As part of a contemplatedmethod, the milled composition may be heated in a microwave blender,e.g., a microwave blender that includes a 75,000 watt microwavetransmitter.

In certain embodiments, a contemplated substantially aerobic organismfree mixed allergen composition has fewer than about 4.5, about 4, about3.5, about 3, about 2.5, about 2, or about 1.5 log CFU/g total aerobicorganisms, and/or the substantially aerobic organism free mixed allergencomposition has fewer than about 1 log CFU/g total coliforms.

In certain embodiments, the substantially aerobic organism free mixedallergen composition has at least about 0.5, about 1, about 1.5, about2, about 2.5, about 3, about 3.5, or about 4 log CFU/g fewer aerobicorganisms than the mixed allergen composition. In certain embodiments,the substantially aerobic organism free mixed allergen composition has asubstantially similar protein structure as the mixed allergencomposition, as measured by SDS-PAGE and/or a substantially similarallergen effect as the mixed allergen composition, as measured by immuneresponse in a patient.

It is contemplated that the bulking agent may comprise a sugar. It iscontemplated that the mixed allergen composition may, e.g., compriseequal parts by protein mass of: at least one nut flour, wherein each nutflour is selected from the group consisting of peanut flour, almondflour, walnut flour, cashew flour, hazelnut flour, pecan flour andpistachio flour; at least one fish powder, wherein each fish powder isselected from the group consisting of cod powder and salmon powder;wheat; and powdered hen's egg or egg white. In certain embodiments, themixed allergen composition may further comprise at least one additionalallergen component, e.g., soy and/or an additional allergen componentselected from oat flour, sesame seed flour, and shrimp flour. In certainembodiments, the mixed allergen composition may further comprise vitaminD. In an exemplary embodiment, the mixed allergen composition comprises30 mg by protein weight shrimp powder; 30 mg by protein weight almondpowder; 30 mg by protein weight wheat; 30 mg by protein weight codpowder; 30 mg by protein weight powdered hen's egg; and 400 IU vitaminD.

Also contemplated herein is a substantially aerobic organism free mixedallergen composition produced by a disclosed method, and a food productcomprising a substantially aerobic organism free mixed allergencomposition produced by a disclosed method.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an SDS-PAGE gel showing a mixed allergen composition (ProductA) following treatment in a WaveMix microwave blender.

FIG. 2 is a line graph depicting the relationship between moisture andwater activity (a_(w)) for the indicated compositions formulated withthe indicated bulking agents.

FIG. 3 is a bar graph depicting levels of background microflora in amixed allergen composition (Product A) at several sampling times duringtreatment in a WaveMix microwave blender. Vertical lines denote standarddeviation (n=3). Dashed line indicates the limit of detection.

FIG. 4 is a bar graph depicting levels of Enterococcus faecium NRRLB-2354 in an artificially-inoculated mixed allergen composition (ProductA) at several sampling times during treatment in a WaveMix microwaveblender. Vertical lines denote standard deviation (Untreated, no water;Untreated, water; and Heated: n=3; Dried: n=10). Dashed line indicatesthe limit of detection.

FIG. 5 is a bar graph depicting water activity of a mixed allergencomposition (Product A) with and without artificially inoculated E.faecium at several sampling times during treatment in the WaveMixmicrowave blender. Vertical lines indicate standard deviation (n=3).Samples within pairs with the same letter were not statisticallydifferent (α=0.05).

DETAILED DESCRIPTION

Disclosed herein are methods for making a mixed allergen composition,e.g., a substantially aerobic organism free mixed allergen composition.A disclosed process may, e.g., comprise one or more of the followingsteps: providing a mixed allergen composition comprising 6 or moreallergens, e.g., 6 to 20 allergens, and a bulking agent, wherein themixed allergen composition comprises at least 6% fat content; millingthe mixed allergen composition in a conical mill to obtain a milledcomposition with substantially consistent particle size; and applyingmicrowaves or radio frequency interference to a mixed allergencomposition, e.g., a milled mixed allergen composition, so that thecomposition is heated to at least 190° F. for at least 30 minutes. Incertain embodiments, the mixed allergen composition comprises at least12% fat content.

The mixed allergen composition may include any allergen or allergencomposition described herein. In certain embodiments, a mixture ofallergens may comprise one, two, or more allergens each independentlyselected from the allergens disclosed in the Examples herein. Forexample, in certain embodiments, a composition may comprise one, two, ormore allergens selected from a group consisting of peanut, soy, almond,cashew, hazelnut, pecan, pistachio, walnut, wheat, oat, milk, egg, cod,salmon, shrimp, and sesame. In certain embodiments, the dry mixture ofallergens includes about 30 mg each of peanut, soy, almond, cashew,hazelnut, pecan, pistachio, walnut, wheat, oat, milk, egg, cod, salmon,shrimp, and sesame. It will be appreciated that the allergenscontemplated herein may each be present as a meal, flour, powder, and/orprotein concentrate.

Contemplated bulking agents may include any bulking agent describedherein. In certain embodiments, the bulking agent comprises a sugar,e.g., sucrose, maltodextrin, or a combination thereof. Without wishingto be bound by theory, it is believed that bulking agents reduce the fatcontent of an allergen mixture to aid in downstream processing, e.g.,milling.

In certain embodiments, a disclosed method comprises milling the mixedallergen composition, e.g., in a conical mill. The milling may, e.g.,comprise using a rotor speed of about 9000 RPM, or may, e.g., furthercomprise applying a vacuum suction through the conical mill. The millingmay, e.g., comprise passing the mixed allergen composition through ascreen with an opening size of about 0.033 inches. Without wishing to bebound by theory, it is believed that milling reduces grittiness andlarge particle size and increases blend homogeneity.

In certain embodiments, a disclosed method comprises applying microwavesor radio frequency interference to a mixed allergen composition, e.g., amilled mixed allergen composition, so that the composition is heated toat least 190° F. for at least 30 minutes, e.g., at least 200° F. for atleast 60 minutes, or at least 230° F. for at least 60 minutes. Incertain embodiments, a mixed allergen composition, e.g., a milled mixedallergen composition, may be heated to no more than 250° F. and/or forno more than 360 or 1000 minutes. In certain embodiments, thecomposition is heated in a microwave blender, e.g., a microwave blenderthat includes a 75,000 watt microwave transmitter. In certainembodiments, a disclosed method comprises applying pressure to a mixedallergen composition, e.g., a milled mixed allergen composition, e.g.,to at least 30 psi for at least 30 minutes. In certain embodiments,applying microwaves or radio frequency interference to the compositionand pressurizing the composition are conducted simultaneously.

In certain embodiments, a disclosed method comprises wetting a mixedallergen composition, e.g., a milled mixed allergen composition. Thecomposition may, e.g., be wetted prior to applying microwaves or radiofrequency interference to heat the composition. In certain embodiments,the composition is wetted to a moisture content of about 6% to about 9%,e.g., 9%, and/or a water activity of about 0.65 to about 0.8, e.g.,about 0.75 to about 0.8.

In certain embodiments, a substantially aerobic organism free mixedallergen composition has fewer than about 4.5, about 4, about 3.5, about3, about 2.5, about 2, or about 1.5 log CFU/g total aerobic organisms.In certain embodiments, a substantially aerobic organism free mixedallergen composition has fewer than about 1 log CFU/g total coliforms.

It is contemplated that, in certain embodiments, a substantially aerobicorganism free mixed allergen composition has fewer aerobic organismsthan a mixed allergen composition that has not been prepared by adisclosed process, e.g., a mixed allergen composition that has not beenheated by microwaves or radio frequency interference, e.g., to at least190° F. for at least 30 minutes, at least 200° F. for at least 60minutes, or at least 230° F. for at least 60 minutes. For example, incertain embodiments, the substantially aerobic organism free mixedallergen composition has at least about 0.5, about 1, about 1.5, about2, about 2.5, about 3, about 3.5, or about 4 log CFU/g fewer totalaerobic organisms than a mixed allergen composition that has not beenprepared by a disclosed process, e.g., a mixed allergen composition thathas not been heated by microwaves or radio frequency interference, e.g.,to at least 190° F. for at least 30 minutes, at least 200° F. for atleast 60 minutes, or at least 230° F. for at least 60 minutes.

It is contemplated that, in certain embodiments, a disclosed method doesnot substantially affect the structure and/or function of the mixedallergen composition. For example, in certain embodiments, asubstantially aerobic organism free mixed allergen composition has asubstantially similar protein structure, as measured by SDS-PAGE, as amixed allergen composition that has not been prepared by a disclosedprocess, e.g., a mixed allergen composition that has not been heated bymicrowaves or radio frequency interference, e.g., to at least 190° F.for at least 30 minutes or at least 230° F. for at least 60 minutes.Similarly, in certain embodiments, a substantially aerobic organism freemixed allergen composition has a substantially similar allergen effect,as measured by immune response in a patient, as a mixed allergencomposition that has not been prepared by a disclosed process, e.g., amixed allergen composition that has not been heated by microwaves orradio frequency interference, e.g., to at least 190° F. for at least 30minutes or at least 230° F. for at least 60 minutes.

As used herein, an “allergenic composition” is understood to mean acomposition that includes one or more different allergens or allergeniccomponents. Allergenic compositions are understood to include “mixedallergen compositions” that include two or more different allergens,where any two given allergens are different if they are distinct fromeach other, e.g., they are compounds described by different chemicalformula or compositions described by different components and/or amountsthereof. The number of different allergens in a composition may vary, asdesired. In certain embodiments, a mixed allergen composition comprises2 or more different allergens, such as 3 or more different allergens, 4or more different allergens, 5 or more different allergens, 6 or moredifferent allergens, 7 or more different allergens, 8 or more differentallergens, 9 or more different allergens, 10 or more differentallergens, 15 or more different allergens, 20 or more differentallergens, 25 or more different allergens, 30 or more differentallergens, 40 or more different allergens, 50 or more differentallergens, 75 or more different allergens, or 100 or more differentallergens. In certain embodiments, a mixed allergen compositioncomprises 100 or fewer different allergens, such as 75 or fewerdifferent allergens, 50 or fewer different allergens, 25 or fewerdifferent allergens, 15 or fewer different allergens, or 10 or fewerdifferent allergens. In certain embodiments, a composition may include 2to 20 different allergens, 2 to 100 different allergens, or 2 to 1000different allergens. In further embodiments, a composition may comprise6 to 20 different allergens. In certain embodiments, a composition mayconsist essentially of 6 to 20 different protein allergens.

Allergens present in an allergenic composition may vary, where in someinstances an allergen present in the composition is one that induces anallergy in a susceptible subject. Allergens include any antigen, oractive derivative thereof, that elicits a specific IgE response.Antigens include any substance that can stimulate the production ofantibodies and can combine specifically with them. Allergens may havelittle or no intrinsic toxicity by themselves, but cause a pathologicalcondition due to their ability to elicit an IgE-associated immuneresponse, and, upon subsequent exposure, due to their ability to elicitIgE- and/or T cell-dependent hypersensitivity reactions. As such, anallergen includes any substance which is capable of stimulating atypical hypersensitivity reaction in atopic subjects. Allergens that maybe present in a given allergenic composition include any substance foundin a variety of different sources, e.g., foods, drugs, perfume, plants,the environment or biological systems (e.g., prokaryotic or eukaryoticcells or viruses), as well as chemical allergens.

It is appreciated that reference to an allergen or an allergeniccomposition may each include a plurality of different proteins as foundin the naturally occurring allergen (either raw or cooked). For example,a provided food product may include a peanut allergen composition (whichwould include substantially all peanut proteins present in e.g.,defatted peanuts, ground peanuts, etc.). As used herein the phrase“complete allergen” refers to all possible antigenic components of agiven food product.

Allergens of interest include nut allergens. Nut allergens are allergensthat include one or more compounds found in nuts, e.g., dry fruits thatinclude an edible kernel or meat enclosed in a woody or leathery shell.Nut allergens of interest include, e.g. peanut allergens, (e.g., rAra h1, rAra h 2, rAra h 3, rAra h 8 PR-10, rAra h 9 LTP, or peanut completeallergen), brazil nut allergens (e.g., rBer e 1, or brazil nut completeallergen), hazelnut or filbert allergens (e.g., rCor a 1 PR-10, rCor a 8LTP, nCor a 9, rCor a 14, or hazel nut complete allergen), walnutallergens (e.g., rJug r 1, rJug r 3 LTP, or walnut complete allergen),cashew allergens (e.g., cashew component allergens, or cashew completeallergen), pistachio allergens (e.g., pistachio component allergens, orpistachio complete allergen), pecan allergens (e.g., pecan componentallergens, or pecan complete allergen), almond allergens (e.g., almondcomponent allergens, or almond complete allergen), or tree nut componentpackage allergens (e.g., one or more allergens from e.g., cashew nut,walnut, hazelnut, or brazil nut).

Allergens of interest include animal allergens. Animal allergens areallergens that include one or more compounds found in animals, includingboth vertebrates and invertebrates. Vertebrate animal allergens that maybe present in an allergenic composition include avian allergens (e.g.,egg allergens, e.g., nGal d 1 Ovomucoid, n Gal d 2 Ovalbumin, nGal d 3Conalbumin, or egg white complete allergen), mammalian allergens (e.g.milk allergens, e.g., nBos d 4 alpha-lactalbumin, nBos d 5beta-lactoglobulin, nBos d 8 Casein, nBos d Lactoferrin, or milkcomplete allergen), or fish allergens (e.g., rCyp c 1, rGad c 1, codcomplete allergen, white fish allergens, or pink fish allergens).Invertebrate animal allergens that may be present in an allergeniccomposition include crustacean allergens (e.g., shrimp allergens, e.g.,rPen a 1 tropomyosin, or shrimp complete allergen), or insect allergens(e.g., bee sting venom allergen, wasp sting venom allergen, or mosquitobite allergen).

Allergens of interest include non-nut plant allergens, i.e., plantallergens that are not nut allergens. Plant allergens are allergens thatinclude one or more compounds found in plants. Plant allergens ofinterest include wheat allergens (e.g., rTri a 19 Omega-5 Gliadin,gliadin wheat, rTri a 14 LTP, or wheat complete allergen), fruitallergens (e.g., kiwi allergens, e.g., rAct d 8 PR-10, or kiwi completeallergen), vegetable allergens (e.g., carrot allergens, or celeryallergens, e.g., rApi g 1.01 PR-10, rPhl p 12, or celery completeallergen), CCD MUXF3 from Bromelain, legume allergens (e.g., soyallergens or chickpea allergens, e.g., rGly m 4 PR-10, nGly m 5Beta-conglycinin, nGly m 6 Glycinin, or soy complete allergen), stonefruit allergens, e.g., f419, f420, f421, f95, f242, o214 rPru p 1 PR-10,rPru p 3 LTP, or stone fruit primary complete allergen), oat allergens(e.g., oat component allergens, or oat complete allergen), or seedallergens (e.g., sesame allergens, e.g., sesame seed componentallergens, or sesame seed complete allergen).

Additional types of allergens that may be present in an allergenic unit,component or composition include, e.g., non-food animal allergens (e.g.,cats or dog fur and dander, cockroach calyx, dust mite excretion), drugallergens (penicillin, sulfonamides, salicylates, local anesthetics),mold spore allergens, latex allergens, metal allergens, or plant pollenallergens (e.g. from grass, e.g., ryegrass or timothy-grass, from weeds,e.g., ragweed, plantago, nettle, Artemisia, vulgaris, chenopodium album,sorrel, or e.g., from trees, e.g., birch alder, hazel, hornbeam,aesculus, willow, poplar, platanus, tilia, or olea).

In certain embodiments, an allergenic composition may comprise one, two,or more allergens selected from a group consisting of cashew, pistachio,walnut, pecan, white fish, pink fish, shrimp, peanut, soy, hazelnut,almond, milk, egg, crab, wheat, and sesame.

In certain embodiments, an allergenic composition may comprise one, two,or more allergens selected from a group consisting of peanut, soy,almond, cashew, hazelnut, pecan, pistachio, walnut, wheat, oat, milk,egg, cod, salmon, shrimp, and sesame.

The amount of a given allergen in an allergenic composition, as desired.In certain embodiments, the amount of a given allergen ranges from about1 to about 15,000 mg, about 5 to about 15,000 mg, about 10 to about10,000 mg, about 15 to about 5,000 mg, about 10 to about 100 mg, orabout 15 to about 100 mg. In certain embodiments, the amount of a givenallergen is about 30 mg. The weight percentage of a given allergen in anallergenic unit, component, or may vary, as desired. In certainembodiments, the weight percentage of a given allergen in an allergenicunit, component, or composition ranges from about 0.1 to about 99.9 wt.%, about 0.1 to about 15 wt. %, about 15 to about 99.9 wt. %, or about25 to about 65 wt. %. The amount of a given allergen in an allergenicunit, component, or composition may be recited by total mass, or byprotein mass, which may vary for a given allergen depending upon theweight percentage of protein in that allergen.

In certain embodiments, if more than one allergen is present in anallergenic composition, e.g., in a mixed allergen composition, any twoof the mixed allergens, or all of the mixed allergens, are present inequal parts, e.g., in a 1:1 ratio, such that each allergen is present inthe composition in equal weight.

A disclosed allergenic composition may include one or more vitamins, asdesired. Vitamins that may be present include. e.g., vitamin A (e.g., inan amount ranging from 1 to 35,000 IU), vitamin C (e.g., in an amountranging from about 1 to about 1,000 mg), vitamin D (e.g., in an amountranging from about 1 to about 4,000 IU, i.e., from about 0.025 to about100 mcg), vitamin E (e.g., in an amount ranging from about 1 to about450 IU), vitamin K (e.g., in an amount ranging from about 1 to about 250mcg), vitamin B-1 (thiamin; e.g., in amount ranging from about 1 toabout 15 mg), vitamin B-2 (riboflavin; e.g., in an amount ranging fromabout 1 to about 17 mg), vitamin B-3 (niacin; e.g., in an amount rangingfrom about 1 to about 200 mg), vitamin B-5 (pantothenic acid; e.g., inan amount ranging from about 1 to about 100 mg), vitamin B-6(pyridoxine; e.g., in an amount ranging from about 1 to about 30 mg),vitamin B-9 (folic acid; e.g., in an amount ranging from about 1 toabout 4,000 mcg), vitamin B-12 (cobalamin; e.g., in an amount rangingfrom about 1 to about 250 mcg), vitamin H (biotin; e.g., in an amountranging from about 1 to about 1,000 mcg) and combinations thereof Incertain embodiments, an allergenic unit, component, or compositioncomprises vitamin D. In certain embodiments, an allergenic unit,component, or composition comprises about 400 IU, i.e., about 10 mcg, ofvitamin D.

It is appreciated that a disclosed allergen or protein may be in theform of a flour, powder, meal, paste, etc. In some embodiments, adisclosed unit or composition comprises about 30 mg protein by weight ofeach specific protein or allergen contained therein, e.g. about 30 mg byprotein weight of an allergenic component each selected as describedherein from peanut, tree nut, cow's milk, soy, egg, fish and shellfish.

Also provided are physiological acceptable compositions that include adisclosed allergenic composition and a physiologically acceptabledelivery vehicle. Disclosed allergenic units, components, orcompositions can be incorporated into a variety of formulations foradministration to a subject. More particularly, a disclosed allergenicunit, component, or composition can be formulated into a physiologicalacceptable composition by combination with appropriate, physiologicallyacceptable carriers or diluents, for example, a vegetable oil. Incertain embodiments, a disclosed allergenic unit, component, orcomposition is designed for oral administration, for example, as foods,tablets, troches, lozenges, aqueous or oily suspensions, dispersiblepowders or granules, emulsions, hard or soft capsules, or syrups orelixirs, gums, etc. Compositions intended for oral use may be preparedaccording to any convenient protocol for the manufacture ofpharmaceutical compositions and such compositions may contain one ormore agents selected from the group consisting of sweetening agents,flavoring agents, coloring agents and preserving agents in order toprovide palatable preparations.

Also provided are allergenic compositions that are food products. Foodproducts of interest include a disclosed allergenic unit, component, orcomposition in combination with a food delivery vehicle. By fooddelivery vehicle is meant a delivery vehicle that is a nourishingsubstance that is eaten, drunk, or otherwise taken into the body tosustain life, provide energy, promote growth, etc. Examples of fooddelivery vehicles or food products of interest include, but are notlimited to: baby or infant formula, baby food (e.g., pureed foodsuitable for infant or toddler consumption), chips, cookies, breads,spreads, creams, yogurts, liquid drinks, chocolate containing products,candies, ice creams, cereals, coffees, pureed food products, etc. Incertain embodiments, the composition is a food supplement.

In certain embodiments, a disclosed allergenic composition may include abulking agent. Exemplary bulking agents include maltodextrin, sucrose,trehalose, trehalose dihydrate, mannitol, lactose, dextrose, fructose,raffinose, or any combination thereof In certain embodiments, thebulking agent comprises maltodextrin, or sucrose, or a combinationthereof. In certain embodiments, the bulking agent comprisesmaltodextrin and sucrose at a weight ratio of about 3:1. In certainembodiments, an allergenic unit, component, or composition may includeexcipients, e.g., a food safe oil, a polysaccharide (e.g., gellan gum),flavoring, and a food safe salt (e.g., dipotassium phosphate).

In certain embodiments an allergenic composition is an aqueoussuspension containing a disclosed allergenic component in admixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients may include suspending agents, for example sodiumcarboxymethyl-cellulose, methylcellulose, hydroxy-propylmethycellulose,sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia;dispersing or wetting agents such as a naturally-occurring phosphatide,for example lecithin, or condensation products of an alkylene oxide withfatty acids, for example polyoxyethylene stearate, or condensationproducts of ethylene oxide with long chain aliphatic alcohols, forexample heptadecaethylene-oxycetanol, or condensation products ofethylene oxide with partial esters derived from fatty acids and ahexitol such as polyoxyethylene sorbitol monooleate, or condensationproducts of ethylene oxide with partial esters derived from fatty acidsand hexitol anhydrides, for example polyethylene sorbitan monooleate.The aqueous suspensions may also contain one or more preservatives, forexample ethyl, or n-propyl, p-hydroxybenzoate, one or more coloringagents, one or more flavoring agents, and one or more sweetening agents,such as sucrose, saccharin or aspartame.

In certain embodiments an allergenic composition is an oily suspensioncontaining an allergenic composition suspended in a vegetable oil, forexample arachis oil, olive oil, sesame oil or coconut oil, or in mineraloil such as liquid paraffin. The oily suspensions may contain athickening agent, for example beeswax, hard paraffin or cetyl alcohol.Sweetening agents such as those set forth above, and flavoring agentsmay be added to provide a palatable oral preparation. These compositionsmay be preserved by the addition of an anti-oxidant such as ascorbicacid.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active ingredient inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents andsuspending agents are exemplified by those already mentioned above.Additional excipients, for example sweetening, flavoring and coloringagents, may also be present.

Disclosed allergenic compositions may also be in the form ofoil-in-water emulsions. The oily phase may be a vegetable oil, forexample olive oil or arachis oil, or a mineral oil, for example liquidparaffin or mixtures of these. Suitable emulsifying agents may benaturally-occurring phosphatides, for example soy bean, lecithin, andesters or partial esters derived from fatty acids and hexitolanhydrides, for example sorbitan monooleate, and condensation productsof the said partial esters with ethylene oxide, for examplepolyoxyethylene sorbitan monooleate. The emulsions may also containsweetening and flavoring agents.

Syrups and elixirs may be formulated with sweetening agents, for exampleglycerol, propylene glycol, sorbitol or sucrose. Such formulations mayalso contain a demulcent, preservative and flavoring and coloringagents. A disclosed composition may be in the form of a sterile aqueousor oleagenous suspension. This suspension may be formulated according tothe known art using those suitable dispersing or wetting agents andsuspending agents which have been mentioned above. The sterilepreparation may also be a sterile solution or suspension in a non-toxicparenterally-acceptable diluent or solvent, for example as a solution in1,3-butane diol. Among the acceptable vehicles and solvents that may beemployed are water, Ringer's solution and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium. For this purpose any bland fixed oilmay be employed including synthetic mono- or diglycerides. In addition,fatty acids such as oleic acid find use in the preparation ofinjectables.

Throughout the description, where apparatus, devices, and systems aredescribed as having, including, or comprising specific components, orwhere processes and methods are described as having, including, orcomprising specific steps, it is contemplated that, additionally, thereare apparatus, devices, and systems that consist essentially of, orconsist of, the recited components, and that there are processes andmethods that consist essentially of, or consist of, the recitedprocessing steps.

The foregoing examples are presented herein for illustrative purposesonly, and should not be construed as limiting in any way.

EXAMPLES Example 1

This example describes the selection of ingredients for inclusion anexemplary dry powder mixed allergen composition containing 16 allergenicingredients (almond, cashew, cod, egg, hazelnut, milk, oat, peanut,pecan, pistachio, salmon, sesame, shrimp, soy, walnut, and wheat)

Ingredients were sourced with primary emphasis on domestic commercialviability, with exceptions made for ingredients that were only availableinternationally. Successful commercial sourcing of multiple options pereach allergenic ingredient led to the development of selection criteriain order to choose the best commercial ingredient to be tested.Attributes screened included maximum protein content and minimum bulkingmaterials, organoleptic attributes, including overall taste, presence ofoff-notes, grittiness, and ingredient solubility. Ingredients ofconsiderably low protein content, or with large proportions of bulkingingredients, were eliminated from contention. Ingredients were tasteddry to determine the presence of off-flavors, as well as assess theingredient's grittiness.

The allergenicity of selected ingredients was also confirmed. Twoinitially selected ingredients, salmon protein and wheat protein, failedprotein gels for allergen confirmation. It is hypothesized that sincethese proteins undergo partial or full hydrolysis in their ingredientprocessing, the amino acid structure is disrupted such that thepolypeptide profile does not result in allergenicity. Despite theprocessability and organoleptic advantages of these ingredients, salmonpowder and wheat gluten powder from different sources replaced the wheatand salmon ingredients in the mixed allergen composition.

Example 2

This example describes the determination of a dry milling process forthe preparation of an exemplary dry powder mixed allergen compositioncontaining 16 allergenic ingredients (almond, cashew, cod, egg,hazelnut, milk, oat, peanut, pecan, pistachio, salmon, sesame, shrimp,soy, walnut, and wheat).

A limitation of dry milling is the fat content of the dry blend. Dryblends with fat content above 6% are difficult to mill withoutconsiderable processing issues, as the fat seeps from the initialingredients and obstructs the milling sieve, thus halting the drymilling process altogether. The blend of the 16 allergenic ingredientsalone exceeded 38% fat, so a large amount of bulking agent was added inorder to feasibly mill the blend.

The 16 protein ingredients were weighed proportionally in order todeliver 30 mg of each protein. Milling of the 16 allergenic ingredientblend with a Quadro Laboratory Scale Dry Mill (FitzMill L1A) at rotorspeeds ranging from 5,000 to 9,000 RPM, and screen sizes of 0.020″,0.033″, or 0.065″, using a blend of sugar and maltodextrin as thebulking agent, allowed for successful milling at a fat content of up to6%.

Milling variables were explored that allowed for a higher fat contentduring the milling process to enable a reduced serving size for the drypowder mixed allergen composition. A Quadro L1A SLS Conical Mill wasused, which has a higher tip speed and is therefore able to achieve amore efficient milling process. A benchtop vacuum (Craftsman) wasattached to the Conical Mill in order to provide suction for the milledpowder to pass through the screen, preventing extended residence time onthe screen, which could lead to the fat melting and clogging the sieve.Sugar was used as a bulking agent which, unlike maltodextrin, has anabrasive crystalline structure which aids in the milling process. Intotal, milling of the 16 allergenic ingredient blend with a Quadro L1ASLS Conical Mill at a rotor speed of 9,000 RPM, and screen size of0.033″, using sugar as the bulking agent, and applying vacuum suction,allowed for successful milling at a fat content of up to 12%.

Other milling processes were unable to successfully mill the mixedallergen composition with the desired fat content. Milling the mixedallergen composition with bulking agent at 15% fat content with aPulvocron and Angled Disintegrator on a 0.5 mm screen resulted inmaterial binding to the screen. Adjusting rotor speed or freezingmaterial in an effort to make it more brittle and resistant to fatmelting and smearing had no impact on the process. Milling the mixedallergen composition with bulking agent at 12% fat content in a RMSRoller Grinder resulted in fat melting, sticking to the rolls andblocking the inter-roller gaps. Milling the mixed allergen compositionwith a Jet Pulverizer at various ratios of protein blend to sugarbulking agent (1:4-1:1.18, corresponding to 6.8%-12% fat content)resulted significant build up in the milling chamber. Lastly, millingthe mixed allergen composition with bulking agent at 20% fat contentwith PowderSize, a jet mill type milling technology, resulting in theblend compacting very quickly in the mill, making milling impossible.

Example 3

This example describes the determination of a sieving process for nutmeals or flours in the preparation of an exemplary dry powder mixedallergen composition containing 16 allergenic ingredients (almond,cashew, cod, egg, hazelnut, milk, oat, peanut, pecan, pistachio, salmon,sesame, shrimp, soy, walnut, and wheat).

Four nut flours which contained larger, more visible particles (pecan,pistachio, almond, and cashew) were passed through a #14 and #18 meshsieve to eliminate the larger particles. Each fraction was tested forprotein content using the Dumas method. The results, depicted in TABLE1, show minimal loss in protein content following each sieving step foreach nut tested. The observed protein content after sieving for each nutfell within the acceptable range for the blend, and variation in proteinquantity found throughout different mesh sizes is in line with normalagricultural variance from batch to batch. These results demonstratethat sieving through a #14 and/or #18 mesh sieve can be used for any ofpecan, pistachio, almond, or cashew flours without impacting proteincontent.

TABLE 1 Percent Protein Change from Nut Sieve Size (%)* Unsieved PecanUnsieved 10.9% N/A Above #14 Mesh 10.6% −2.75% Between #14 and #18 Mesh10.7% −1.83% Below #18 Mesh 11.4% +4.59% Pistachio Unsieved 21.9% N/AAbove #14 Mesh 21.9%  0.00% Between #14 and #18 Mesh 21.9%  0.00% Below#18 Mesh 22.1% +0.91% Almond Unsieved 22.2% N/A Above #14 Mesh 22.2% 0.00% Below #18 Mesh 22.2%  0.00% Cashew Unsieved 17.5% N/A Above #14Mesh 17.7% +1.14% Below #18 Mesh 17.5%  0.00%

Three of the four nuts flours (pecan, pistachio, and almond) showed asignificant benefit due to sieving, e.g. with reduced particle size.Accordingly, pecan and almond flour were sieved through the #14 mesh,and pistachio was sieved through the #18 mesh. Using finer sieves wasnot practical due to the high fat content of the nuts leading toclogging of finer mesh. While sieving did not significantly benefit thecashew flour, it was determined that the cashew flour was fine enoughand light-colored enough, and already had a fine particle sizedistribution, and therefore was suitable to incorporate into theallergen blend without sieving.

Example 4

This example describes the evaluation of the vitamin D content of anexemplary dry powder mixed allergen composition containing 16 allergenicingredients (almond, cashew, cod, egg, hazelnut, milk, oat, peanut,pecan, pistachio, salmon, sesame, shrimp, soy, walnut, and wheat)throughout various exemplary processing steps to determine the impact ofthese steps on vitamin D content and blend homogeneity in general.

First, a Ro-Tap test was designed to simulate mechanical stress andextreme vibration that may occur in a stick packaging machine hopperduring filling. A batch of exemplary dry mixed allergen compositioncontaining 16 allergenic ingredients was blended with vitamin D, flavorand sugar, mixed, loaded in a cylinder, secured on a Ro-Tap and shakenfor 30 minutes. Vitamin D content was tested before the Ro-Tap process,as well as after the Ro-Tap process, from sampling points at the top andthe bottom of the cylinder of product. Vitamin D content was assayed byLCMS. The initial vitamin D content in the mixed allergen formulationbefore the Ro-Tap process was 10.5 mcg/5 g composition. After the Ro-Tapprocess, a top layer sample contained 9.45 mcg vitamin D/5 gcomposition, while the bottom layer was tested at 10.3 mcg vitamin D/5 gcomposition. The results of this experiment suggest that while somevitamin D content differences were measurable in the Ro-Tap product, theextent of any separation is low: in the top layer, vitamin D wasdepleted by ˜10% as a result of the shaking treatment, while the bottomlayer did not show any significant increase in vitamin D concentration.

A second experiment was carried out at a pilot plant scale using aplacebo formulation containing flax seed meal as a stand-in for a mixedallergen composition, as well as sugar, flavor, and vitamin D. Aproduction and packaging process was carried out. Briefly, theingredients were pre-blended in a WaveMix microwave blender withoutmicrowave heating, the blended ingredients were then heated in theWaveMix microwave blender up to 230° F. for one hour, the product wasdischarged into poly-lined pails and allowed to cool, and the productwas then packaged into individual 5 g stick packs.

Blend homogeneity and vitamin D content were evaluated through theproduction and packaging process. Due to the flow characteristics of thedry blended formulation, a vibration component was added to the hopperfeeding into the stick pack filler, which prompted concerns over vitaminD settling out in the hopper. Stick-pack filling was carried out on twoseparate dates, and sampling was carried out at the beginning, middleand end of the run each day. For each sampling point, sets of threestick packs were consolidated for sampling, and the tests were performedin triplicate. Results showed an average of 9.78 mcg/5 g at thebeginning, 9.32 mcg/5 g in the middle, and 9.57 mcg/5 g at the end forthe first day of packaging, and an average of 9.73 mcg/5 g at thebeginning, 9.67 mcg/5 g in the middle, and 10.02 mcg/5 g at the end forthe second day of packaging. Statistical analysis of data showed nosignificant or systematic vitamin D settling observed for a placeboformulation production carried out under typical plant operatingconditions (vibrating hopper, stick pack filler, etc.). However, asthese results consistently showed slightly lower vitamin D content thanthe target dose of 10 mcg/5 g present in the initial composition, a 20%vitamin D overage was used, where indicated, to ensure that each servinghits the minimum targeted dose of 10 mcg/5 g.

A third experiment was carried out at the pilot plant scale using themixed allergen composition containing 16 allergenic ingredients, sugar,flavor, flow agent and vitamin D. Briefly, the ingredients werepre-blended in a WaveMix microwave blender without microwave heating,the blended ingredients were then heated in the WaveMix microwaveblender up to 230° F. for one hour, the product was discharged intopoly-lined pails and allowed to cool, and the product was then packagedinto individual 5 g stick packs.

Blend homogeneity and vitamin D content were evaluated through theproduction and packaging process. Sampling was carried out at thebeginning, middle and end of the stick-pack filling run. For eachsampling point vitamin D testing was performed in six replicates, inorder to increase the power of the statistical analysis. Results showedan average of 10.51 mcg/5 g at the beginning, 11.36 mcg/5 g in themiddle, and 11.08 mcg/5 g at the end of the packaging run. Data analysisshowed no statistically significant differences in vitamin Dconcentration throughout a full mixed allergen formulation productioncarried out under typical plant operating conditions (vibrating hopper,stick pack filler, etc.). These results demonstrate that there is nosettling out of vitamin D in the final mixed allergen formulationthroughout a full production and packaging process.

In order to further ensure vitamin D homogeneity in a mixed allergencomposition, the following process was developed. The 16 allergenicingredients (almond, cashew, cod, egg, hazelnut, milk, oat, peanut,pecan, pistachio, salmon, sesame, shrimp, soy, walnut, and wheat) weredry blended together, resulting in a high overall fat content blend.Vitamin D was added and dry blended with the 16 allergenic ingredientblend, so that vitamin D ingredient particles can stick to the higherfat, oily nut meal particles, helping to disperse the vitamin D in thefinished product matrix. This process further prevents separation orsettling of vitamin D in the finished product.

Example 5

The formulation of an exemplary dry powdered mixed allergen compositionresulting from the processes described in Examples 1-2 is depicted inTABLE 2. This composition is hereafter referred to as Product A.

TABLE 2 Ingredient % Dry Blend Peanut Flour 1.107 Soy Protein Isolate0.66 Blanched Almond Flour 2.64 Cashew Flour 3.14 Hazelnut Flour 1.95Pecan Meal 6.28 Pistachio Meal 2.69 Walnut Flour 1.33 Wheat GlutenPowder 0.74 Oat Protein Powder 1.05 Milk Protein Isolate 0.677 DriedWhole Eggs 1.15 Codfish Powder 0.75 Salmon Powder 1.55 Shrimp Powder0.88 Sesame Seed Flour 0.99 Dry Vitamin D3 100 SD/S 0.08 Natural MaskingFlavor 9.45 Extra Fine Granulated Sugar 60.93 Rice concentrate 2.01Total 100

The formulation of an additional exemplary dry powdered composition,including brown flax seed, golden flax seed, caramel color, naturalmasking flavor, sugar (bulking agent), rice concentrate (flow aidingredient), and vitamin D is depicted in TABLE 3. This composition ishereafter referred to as Product B.

TABLE 3 Ingredient % Dry Blend Brown Flax Seed 18.53 Gold Flax Seed 8.79Caramel Color 0.21 Dry Vitamin D3 100 SD/S 0.08 Natural Masking Flavor9.45 Extra Fine Granulated Sugar 60.93 Rice concentrate 2.01 Total 100

The formulation of an additional exemplary dry powdered mixed allergencomposition resulting from the processes described in Examples 1-2 isdepicted in TABLE 4. This composition is hereafter referred to asProduct C. Product C was based on the formulation of Product A, andincorporates improvements related to improving the flow of the drypowder product.

TABLE 4 Ingredient % Dry Blend Peanut Flour 1.13 Soy Protein Isolate0.70 Blanched Almond Flour 2.80 Cashew Flour 3.33 Hazelnut Flour 2.07Pecan Meal 6.67 Pistachio Meal 2.86 Walnut Flour 1.41 Wheat GlutenPowder 0.79 Oat Protein Powder 1.11 Milk Protein Isolate 0.71 DriedWhole Eggs 1.22 Codfish Powder 0.80 Salmon Powder 1.65 Shrimp Powder0.83 Sesame Seed Flour 1.05 Dry Vitamin D3 100 SD/S 0.08 Natural MaskingFlavor 9.40 Extra Fine Granulated Sugar 59.88 Silicon Dioxide Sipernat50 1.50 Total 100

The formulation of an additional exemplary dry powdered mixed allergencomposition resulting from the processes described in Examples 1-2 isdepicted in TABLE 5. This composition is hereafter referred to asProduct D.

TABLE 5 Ingredient % Dry Blend Peanut Flour 1.13 Soy Protein Isolate0.70 Blanched Almond Flour 2.80 Cashew Flour 3.33 Hazelnut Flour 2.07Pecan Meal 6.67 Pistachio Meal 2.86 Walnut Flour 1.41 Wheat GlutenPowder 0.79 Oat Protein Powder 1.11 Milk Protein Isolate 0.71 DriedWhole Eggs 1.22 Codfish Powder 0.80 Salmon Powder 1.65 Shrimp Powder0.94 Sesame Seed Flour 1.05 Dry Vitamin D3 100 SD/S 0.08 Natural MaskingFlavor 9.40 Sweetener 0.12 Maltodextrin 45.84 Extra Fine GranulatedSugar 15.32 Total 100.00

The formulation of an additional exemplary dry powdered mixed allergencomposition resulting from the processes described in Examples 1-3 isdepicted in TABLE 6. This composition is hereafter referred to asProduct E. Product E was based on the formulation of Product A modifiedwith the goal of reducing the sugar content in the mixed allergencomposition. 75% of the extra fine granulated sugar was replaced with anagglomerated maltodextrin to improve visual characteristics of thefinished product, and sweetener was added to provide sweetness lost dueto replacement of sugar by maltodextrin. Blanched almond flour, pecanand pistachio meals were sieved in order to remove large particles fromthe finished product, therefore visually improving the product.

TABLE 6 Ingredient % Dry Blend Peanut Flour 1.13 Soy Protein Isolate0.70 Blanched Almond Flour, sieved 2.80 Cashew Flour 3.33 Hazelnut Flour2.07 Pecan Meal, sieved 6.67 Pistachio Meal, sieved 2.86 Walnut Flour1.41 Wheat Gluten Powder 0.79 Oat Protein Powder 1.11 Milk ProteinIsolate 0.71 Dried Whole Eggs 1.22 Codfish Powder 0.80 Salmon Powder1.65 Shrimp Powder 0.83 Sesame Seed Flour 1.05 Dry Vitamin D3 100 SD/S0.08 Natural Masking Flavor 9.40 Sweetener 0.12 Maltodextrin 45.84 ExtraFine Granulated Sugar 15.43 Total 100

The formulation of an additional exemplary dry powdered mixed allergencomposition resulting from the processes described in Examples 1-4 isdepicted in TABLE 7. This composition is hereafter referred to asProduct F. Product F was based on the formulation of Product C. ProductF was modified to remove maltodextrin due to processing challenges.Sweetener was not included in the formulation. Additionally, a 20%vitamin D overage was included based on results described in Example 4,to ensure the final composition after processing contains the targetedvitamin D dose.

TABLE 7 Ingredient % Dry Blend Peanut Flour 1.13 Soy Protein Isolate0.70 Blanched Almond Flour, sieved 2.80 Cashew Flour 3.33 Hazelnut Flour2.07 Pecan Meal, sieved 6.67 Pistachio Meal, sieved 2.86 Walnut Flour1.41 Wheat Gluten Powder 0.79 Oat Protein Powder 1.11 Milk ProteinIsolate 0.71 Dried Whole Eggs 1.22 Codfish Powder 0.80 Salmon Powder1.65 Shrimp Powder 0.83 Sesame Seed Flour 1.05 Dry Vitamin D3 100 SD/S0.10 Natural Masking Flavor 9.40 Extra Fine Granulated Sugar 59.87Silicon Dioxide Sipernat 50 1.50 Total 100

The formulation of an exemplary dry powdered mixed allergen compositionresulting from the processes described in Examples 1-2 is depicted inTABLE 8. This composition is hereafter referred to as Product G.

TABLE 8 Ingredient % Dry Blend Peanut Flour 1.31 Soy Protein Isolate0.74 Blanched Almond Flour 2.99 Cashew Flour 3.56 Hazelnut Flour 2.21Pecan Meal 7.12 Pistachio Meal 3.05 Walnut Flour 1.51 Wheat GlutenPowder 0.84 Oat Protein Powder 1.19 Milk Protein Isolate 0.75 DriedWhole Eggs 1.31 Codfish Powder 0.85 Salmon Powder 1.76 Shrimp Powder1.00 Sesame Seed Flour 1.12 Dry Vitamin D3 100 SD/S 0.09 Natural MaskingFlavor 9.46 Extra Fine Granulated Sugar 57.11 Rice concentrate 2.03Total 100

Example 6

This example describes an exemplary microbial reduction treatment for adry powdered product using a microwave blender.

Product B (as described in Example 5) was subjected to a treatment of230° F./110° C. for 60 minutes in a WaveMix microwave blender, and thetotal aerobic organisms were measured before and after treatment.Additionally, Product B was inoculated with a population of an indicatorvegetative organism, Enterococcus faecium NRRL B-2354, prior toundergoing treatment in the microwave blender, and the total E. faeciumwas measured before and after heat treatment for inoculated Product B.E. faecium has been used as a non-pathogenic Salmonella surrogate forthermal lethality studies carried out on dried products because it isrelatively heat resistant at lower water activities. In the Examplesherein, unless indicated otherwise, the WaveMix microwave blender usedwas a Model No. SPU-2436 with a 75,000 watt microwave transmitter.

Samples were diluted and spread onto a petri dish of general recoverymedia to measure colony-forming units per gram of product (CFU/g). Foruninoculated Product, the limit of detection for the method was 1 CFU/gfor a 1:10 dilution. For inoculated Product, the limit of detection wasset to the first serial dilution at which no background microfloragrowth was observed in the uninoculated samples.

The total aerobic organisms for uninoculated Product B before and aftertreatment in the WaveMix microwave blender and the log reduction due totreatment are depicted in TABLE 9. The total E. faecium for theinoculated Product B before and after treatment in the WaveMix microwaveblender and the log reduction due to treatment are depicted in TABLE 10.Throughout the specification, Avg±SD indicates average±standarddeviation. Log reduction was calculated by subtracting the counts fromeach replicate after treatment from the average count before treatment.

TABLE 9 Total Counts (log CFU/g) Target Before After Log OrganismReplicate Treatment Treatment Reduction Total aerobic A 5.0 3.7 1.7organisms B 5.5 3.9 1.5 C 5.6 3.8 1.6 Avg ± SD 5.4 ± 0.3 3.8 ± 0.1 1.6 ±0.1

TABLE 10 Total Counts (log CFU/g) Target Before After Log OrganismReplicate Treatment Treatment Reduction E. faecium A 7.4 <4.0¹ >3.5 B7.4 <4.0¹ >3.5 C 7.5 <4.0¹ >3.5 D — <4.0¹ >3.5 E — <4.0¹ >3.5 F —<4.0¹ >3.5 G — <4.0¹ >3.5 H — <4.0¹ >3.5 I — <4.0¹ >3.5 J — <4.0¹ >3.5Avg ± SD 7.5 ± 0.0 <4.0¹ >3.5 ¹Below the limit of detection of 4.0 logCFU/g

Uninoculated Product B had a 1-2 log reduction in the backgroundmicroflora after treatment in the WaveMix microwave blender, whileinoculated Product B had a greater than 3.5 log reduction in E. faeciumdue to treatment in the WaveMix microwave blender.

Product A (as described in Example 5) was subjected to a treatment of230° F./110° C. for 60 minutes in a WaveMix microwave blender, and thetotal aerobic organisms were measured before and after treatment. Thecounts before and after treatment in the WaveMix microwave blender andthe log reduction due to treatment are depicted in TABLE 11. Logreduction was calculated by subtracting the counts from each replicateafter treatment from the average count before treatment.

TABLE 11 Total Counts (log CFU/g) Target Before After Log OrganismReplicate Treatment Treatment Reduction Total aerobic A 2.7 2.9 Noreduction organisms B 3.6 3.0 C 3.4 2.8 Avg ± SD 3.2 ± 0.5 2.9 ± 0.1

No reduction in total aerobic organisms was observed in Product A aftertreatment in the WaveMix microwave blender.

Following treatment, SDS-PAGE testing of Product A was performed asfollows. Product A was solubilized in reducing Laemmli buffer to aconcentration of 1 mg protein/ml. Samples were mixed, heated at 95°C.-100° C., centrifuged, and loaded onto an SDS-PAGE gel. The gel wasrun at 100V for 80 to 90 minutes. Following completion, the gel wasfixed, stained, destained and imaged. As shown in FIG. 1 , SDS-PAGEtesting revealed no damage to the protein structure of the Product Acomponents following treatment in the WaveMix microwave blender.

In summary, microbial reduction treatment in a WaveMix microwave blenderresulted in a 1-2 log reduction of background microflora for Product B,and a >3.5 log reduction in E. faecium when Product B was inoculatedwith E. faecium prior to treatment. No reduction in total microbialcontent was observed in the background microflora of Product A aftertreatment in a WaveMix microwave blender. These results suggest that,under certain conditions, microwave blender treatment may substantiallyreduce population levels of vegetative cells in a dry powderedcomposition.

Example 7

This example describes an exemplary microbial reduction treatment for adry powdered product using a microwave blender to treat a wettedproduct.

As water activity (a_(w)) may influence the time required for thermalreduction of target microorganisms, the relationship between moistureand water activity was determined for exemplary dry powdered products.Products A and B were prepared as described in Example 5, except withvarying bulking agents as described below. Product B was formulated at15% total fat content using powdered sugar as a bulking agent, andProduct A was formulated at 15% total fat content using each ofgranulated sugar, maltodextrin, and powdered sugar as bulking agents.700 g of each Product was mixed for five minutes in a five quartKitchenAid mixer bowl using a whisk. Water was added stepwise underagitation for an additional 8 minutes in the KitchenAid mixer, whilemoisture was measured using a Computrac 1000XL at 135° C. and wateractivity (a_(w)) measured using a Rotronic HygroLab. The relationshipbetween moisture and water activity for the indicated formulations isdepicted in FIG. 2 .

Product B formulated with powdered sugar had systematically higher a_(w)values than Product A, consistently achieving a_(w)22 0.8 at moisturelevels ≥7.5%.

For Product A, granulated sugar and powdered sugar were more effectivebulking agents than maltodextrin, as Product A formulated withmaltodextrin required more than three times the level of moisture thanpowdered sugar in order to reach an equivalent a_(w)>0.7. The particlesize of sugar used in the experiment did not have a significant effecton the moisture-a_(w) relationship, as formulations with granulated andpowdered sugar bulking agents behaved similarly.

In summary, for the products tested, sugar was the best performingbulking agent by allowing formulation of systems with high wateractivity at the lowest moisture content values (7.5-10%), which may leadto higher lethality during microwave blender processing. Together, theseresults suggest that microbial reduction treatment for a mixed allergencomposition, e.g., Product A, using a microwave blender may be enhancedby moistening to a 6-9% final moisture content (corresponding to ana_(w) of 0.65-0.8), and using powdered sugar as a bulking agent.

Based on these results, microbial reduction treatment using a microwaveblender was tested on a wetted powdered product. Product G (as describedin Example 5) was wetted and subjected to a treatment of 200° F./93° C.for 60 minutes in a WaveMix microwave blender, and then dried in theblender. Total aerobic organisms were measured before water addition andheat treatment, after water addition and before heat treatment, afterheat treatment and before drying, and, finally, after drying.Additionally, Product G was inoculated with a population of an indicatorvegetative organism, E. faecium, prior to wetting and heat treatment,and the total E. faecium was measured at each sampling time for theinoculated Product G.

Samples were diluted and spread onto a petri dish of general recoverymedia to measure colony-forming units per gram of product (CFU/g). Foruninoculated Product, the limit of detection for the method was 1 CFU/gfor a 1:10 dilution. For inoculated Product, the limit of detection wasset to the first serial dilution at which no background microfloragrowth was observed in the uninoculated samples.

The total aerobic organisms for the uninoculated Product G at theindicated times and the log reduction due to heat treatment are depictedin TABLE 12 and FIG. 3 . The total E. faecium for the inoculated ProductG at the indicated times and the log reduction due to heat treatment aredepicted in TABLE 13 and FIG. 4 . Log reduction was calculated bysubtracting the counts from each replicate after treatment from theaverage count before treatment.

TABLE 12 Total Counts (log CFU/g) Target Untreated, Untreated, OrganismReplicate No Water Water Heated Dried Total aerobic A 5.1 2.1 2.2 2.6Organisms B 2.7 2.5 2.1 2.4 C 2.4 2.5 2.2 2.3 Avg ± SD 3.4 ± 1.5 2.4 ±0.2 2.2 ± 0.0 2.4 ± 0.2 Log No No No Reduction Reduction ReductionReduction

TABLE 13 Total Counts (log CFU/g) Target Untreated, Untreated, OrganismReplicate No Water Water Heated Dried E. faecium A 7.4 6.7 3.5 4.5 B 7.46.7 3.1 4.5 C 7.7 7.1 4.2 4.3 D — — — 4.7 E — — — 4.5 F — — — 4.5 G — —— 4.7 H — — — 4.3 I — — — 5.2 J — — — 4.6 Avg ± SD 7.5 ± 0.1 6.8 ± 0.23.6 ± 0.6 4.4 ± 0.1 Log No 3.9 ± 0.6 2.9 ± 0.3 Reduction Reduction

No reduction in the level of background microflora of the uninoculatedProduct G was observed after treatment in the WaveMix microwave blender,while a 3-4 log reduction in E. faecium was observed for the inoculatedProduct G after treatment in the WaveMix microwave blender.

Water activity and moisture content were also measured throughout thetreatment process using a Rotronic HygroLab or AquaLab 4TE and Computrac1000XL, respectively. Water activity for inoculated and uninoculatedProduct G at the indicated time points is depicted in FIG. 5 , and wateractivity and moisture content of inoculated and uninoculated Product Gat the indicated time points is depicted in TABLE 14.

TABLE 14 Results at Each Sampling Time Untreated, Untreated, ProductAnalysis Replicate No Water Water Heated Dried Inoculated Water A 0.2700.623 0.482 0.104 Activity B 0.271 0.628 0.471 0.110 C 0.272 0.633 0.3830.140 Avg ± SD 0.271 ± 0.001 0.628 ± 0.005 0.445 ± 0.054 0.118 ± 0.019Moisture A 2.73% 5.47% 4.11% 2.32% Content B 2.86% 4.94% 3.23% 1.89% C2.85% 5.26% 2.85% 2.46% Avg ± SD    2.81 ± 0.07%    5.22 ± 0.27%    3.40± 0.65%    2.22 ± 0.30% Uninoculated Water A 0.274 0.687 0.474 0.136Activity B 0.272 0.675 0.494 0.146 C 0.275 0.684 0.487 0.151 Avg ± SD0.274 ± 0.001 0.682 ± 0.006 0.485 ± 0.010 0.144 ± 0.007 Moisture A 1.61%6.72% 4.52% 1.69% Content

In summary, microbial reduction treatment in a WaveMix microwave blenderresulted in no reduction in background microflora for an exemplary mixedallergen composition; however, treatment resulted in a 3-4 log reductionin E. faecium in mixed allergen composition inoculated with E. faeciumprior to treatment. These results suggest that, under certainconditions, microwave blender treatment may substantially reducepopulation levels of vegetative cells in a mixed allergen composition.

Example 8

This example describes an exemplary microbial reduction treatment for adry powdered product using a pressurized heat treatment.

Products A and B are formulated with 12% fat and 6-9% final moisturecontent with powdered sugar as a bulking agent, and subjected totreatment at 240° F. for 30 minutes at 30 psi and at 200° F. for 60minutes at atmospheric pressure in a microwave blender. The pressurizedtreatment allows for higher temperatures without boiling water. Eachprocess is carried out in a closed system environment in order toprevent moisture loss and large shifts in a_(w) during the heatingprocess.

Example 9

This example describes an exemplary microbial reduction treatment for adry powdered product using Radio Frequency Interference (RFI).

Radio Frequency (RF) is a long-wave length, non-ionizing form ofelectrical energy. RF's long-wavelength energy have a superior depth ofpenetration relative to microwaves, allowing for a rapid, uniform andvolumetric heating of a product, simultaneously throughout the entirethickness. Radio frequency can also be carried out both in bulk as wellas in packaged material, provided the packaging does not contain a foilcomponent or carbon black markings.

RFI microbial reduction treatment was carried out an exemplary drypowdered mixed allergen composition (Product A as described in Example5).

Bulk quantities of Product A (approximately 2 pounds) were treated at250° F., 225° F., 215° F., 205° F., 195° F., and 190° F. in a Macrowave™radio frequency pasteurization unit.

Microbiological testing was carried out before the RFI treatment, afterthe lowest heat treatment temperature (190° F.) and after the highesttemperature treatment (250° F.). Total coliforms, and total aerobicorganisms were determined by spreading diluted samples onto a petri dishof recovery media to measure colony-forming units per gram of product(CFU/g). The results are depicted in TABLE 15. The RFI process reducedthe aerobic plate count, with the 190° F. treatment resulting in a 0.7log reduction, and the 250° F. treatment resulting in a 1.8 logreduction. The RFI process also reduced the total coliforms, with the190° F. treatment resulting in a 0.5 log reduction, and the 250° F.treatment resulting in a >1.2 log reduction. The 250° F. treatmentresulted in a population of coliforms that was beneath the detectionthreshold.

TABLE 15 Before Low High Test Treatment Temperature Temperature AerobicPlate Count 3.8 log 3.1 log 2.0 log (APC) CFU/g CFU/g CFU/g APC logreduction — 0.7 log 1.8 log Total Coliforms 2.2 log 1.7 log  <1 logCFU/g CFU/g CFU/g

These results suggest that, under certain conditions, RFI treatment mayreduce microorganism population in a mixed allergen composition.

Example 10

This example describes an exemplary thermal treatment for a dry powderedproduct.

Product D (as described in Example 5) was placed into a Kenwood mixerequipped with an induction heating system, and heat treated at 230° F.for 60 minutes under continuous mixing. The Kenwood mixer equipped withan induction heating system mimics the effects of a microwave blender,such as the WaveMix microwave blender, at the bench scale. Although aplastic splash-guard was used as a cover, the system was not closed andas a result moisture could evaporate throughout the treatment.

A comparison of the properties of the heat treated product versus anun-heated control was carried out. The heat treated product was testedas both as a powder, as well as a powder mixed in applesauce. Thesweetness, flavor and aroma were not impacted by heat treatment, whilethe heat treated sample was slightly darker. As a dry powder, the heattreated sample clumped slightly more in the mouth than untreated sample.The heat treated product also clumped slightly upon mixing inapplesauce, however, after 2-3 minutes the clumps disappeared and theblend looked homogeneous.

These results suggest that the heat treatment of a mixed allergencomposition, e.g., Product D, does not substantially influence taste,color, or clumping of the mixed allergen composition.

Example 11

This example describes an exemplary microbial reduction treatment for adry powdered product using a microwave blender.

Product C (as described in Example 5) was processed in a pilot plantsetting using the following process. All ingredients were weighed andpre-blended in a 10 cubic foot WaveMix microwave blender for 5 minutes.Vitamin D was added and the composition was further blended for 5minutes to fully disperse the fine powder and attach it to the fatty nutparticles in the 16 protein ingredient blend. Sugar and natural maskingflavor were added and the composition was further blended for 5 minutesuntil homogeneous. After preparing the homogeneous blend, heat treatmentwas carried out in the microwave blender in two phases. In a firstheating phase, the microwave power was set at 35 kW and the blender wasset at 30 rpm, with the goal of increasing product temperature to 230°F. A typical duration for this stage was 18-21 minutes. In a secondholding phase, the product was maintained at 230° F. for 60 minutesunder constant agitation at 30 rpm to prevent localized overheating. Atthe end of the WaveMix process, the product was discharged hot intolined pails and allowed to cool to room temperature for up to 48 hours.After cooling, the product was filled into stick-packs, at a targetweight of 5-5.4 g per stick-pack.

Product C (as described in Example 5) was subjected to a treatment of230° F./110° C. for 60 minutes in a WaveMix microwave blender, and thetotal aerobic organisms were measured before and after treatment.Additionally, Product C was inoculated with E. faecium NRRL B-2354,prior to undergoing treatment in the microwave blender, and the total E.faecium was measured before and after heat treatment for inoculatedProduct C.

Samples were diluted and spread onto a petri dish of general recoverymedia to measure colony-forming units per gram of product (CFU/g). Foruninoculated and inoculated Product C, the limit of detection for themethod was 1 CFU/g for a 1:10 dilution.

The total aerobic organisms for uninoculated Product C before and aftertreatment in the WaveMix microwave blender and the log reduction due totreatment are depicted in TABLE 16. The total E. faecium for theinoculated Product C before and after treatment in the WaveMix microwaveblender and the log reduction due to treatment are depicted in TABLE 17.Throughout the specification, n/d indicates not determined. Logreduction was calculated by subtracting the counts from each replicateafter treatment from the average count before treatment.

TABLE 16 Total Counts (log CFU/g) Before After Organism ReplicateTreatment Treatment Log Reduction Background A 2.8 1.4 1.5 microflora B2.4 1.8 1.1 C n/d 1.6 1.3 D 3.5 <1.0¹ >1.9 E 2.9 <1.0¹ >1.9 Avg ± SD 2.9± 0.5 1.4 ± 0.4 1.5 ± 0.4 ¹Below the limit of detection

TABLE 17 Trial 1 Trial 2 Total Counts Total Counts (log CFU/g) (logCFU/g) Before After Log Before After Log Organism Replicate TreatmentTreatment Reduction Treatment Treatment Reduction E. A 6.7 <1.0¹ >5.66.8 <1.0¹ >5.8 faecium B 6.7 <1.0¹ >5.6 6.9 1.5 5.3 C 6.7 <1.0¹ >5.6 6.7<1.0¹ >5.8 D 6.9 <1.0¹ >5.6 6.7 1.2 5.6 E 6.9 n/d >5.6 6.8 2.5 4.3 F 6.61.6 5.0 6.9 1.9 4.9 G 6.4 <1.0¹ >5.6 6.9 1.2 5.6 H 6.5 1.6 5.0 6.6<1.0¹ >5.8 I 6.4 1.2 5.4 6.6 <1.0¹ >5.8 J 6.5 2.0 4.6 6.6 2.2 4.6 K —<1.0¹ >5.6 — <1.0¹ >5.8 L — <1.0¹ >5.6 — <1.0¹ >5.8 M — 2.2 4.4 —<1.0¹ >5.8 N — 2.1 4.5 — <1.0¹ >5.8 O — 1.5 5.1 — <1.0¹ >5.8 P — 1.4 5.2— <1.0¹ >5.8 Q — 1.6 5.0 — 1.4 5.4 R — 1.2 5.4 — 1.8 5.0 S — <1.0¹ >5.6— <1.0¹ >5.8 T — 2.9 3.7 — <1.0¹ >5.8 Avg ± SD 6.6 ± 0.2 1.4 ± 0.5 5.2 ±0.5 6.8 ± 0.1 1.3 ± 0.5 5.5 ± 0.5 ¹Below the limit of detection

A 1-2 log reduction in the level of background microflora of theuninoculated Product C was observed after treatment in the WaveMixmicrowave blender, while a 5 log reduction in E. faecium was observedfor the inoculated Product C after treatment in the WaveMix microwaveblender.

Water activity of Product C was also measured on-site immediately beforeand after the treatment process and before enumeration in the lab usingan AquaLab 4TE. Water activity for inoculated and uninoculated Product Cat the indicated time points is depicted in TABLE 18.

TABLE 18 Water Activity Trial 1 Trial 2 Trial 3 Sampling Before AfterBefore After Before After Time Replicate Treatment Treatment TreatmentTreatment Treatment Treatment Utah A 0.326 <0.025 0.331 <0.025 0.322<0.025 facility Livermore A 0.327 0.083 0.348 0.042 0.343 0.052 facilityB 0.341 0.056 0.360 0.070 0.346 0.036 C 0.342 0.083 0.366 0.057 0.3390.038 Avg ± SD¹ 0.337 ± 0.008 0.074 ± 0.016 0.358 ± 0.009 0.056 ± 0.0140.343 ± 0.004 0.042 ± 0.009

In summary, microbial reduction treatment in a WaveMix microwave blenderresulted in a 1-2 log reduction in background microflora for anexemplary mixed allergen composition; however, treatment resulted in a5-log reduction in E. faecium in the mixed allergen compositioninoculated with E. faecium prior to treatment. These results suggestthat, under certain conditions, microwave blender treatment maysubstantially reduce population levels of vegetative cells in a mixedallergen composition.

INCORPORATION BY REFERENCE

All publications and patents mentioned herein, including those itemslisted below, are hereby incorporated by reference in their entirety forall purposes as if each individual publication or patent wasspecifically and individually incorporated by reference. In case ofconflict, the present application, including any definitions herein,will control.

EQUIVALENTS

While specific embodiments of the subject invention have been discussed,the above specification is illustrative and not restrictive. Manyvariations of the invention will become apparent to those skilled in theart upon review of this specification. The full scope of the inventionshould be determined by reference to the claims, along with their fullscope of equivalents, and the specification, along with such variations.

Unless otherwise indicated, all numbers expressing quantities ofingredients, reaction conditions, and so forth used in the specificationand claims are to be understood as being modified in all instances bythe term “about.” Accordingly, unless indicated to the contrary, thenumerical parameters set forth in this specification and attached claimsare approximations that may vary depending upon the desired propertiessought to be obtained by the present invention.

1. A method of making a substantially aerobic organism free mixedallergen composition, comprising: providing a mixed allergen compositioncomprising 6 to 20 allergens and a bulking agent, wherein the mixedallergen composition comprises at least 6% fat content; milling themixed allergen composition in a conical mill to obtain a milledcomposition with substantially consistent particle size; and applyingmicrowaves or radio frequency interference to the milled composition sothat the milled composition is heated to at least 190° F. for at least30 minutes, thereby to obtain the substantially aerobic organism freemixed allergen composition.
 2. The method of claim 1, wherein the mixedallergen composition comprises at least 12% fat content.
 3. The methodof claim 1, wherein milling the mixed allergen composition comprisesusing a rotor speed of about 9000 RPM.
 4. The method of claim 1, whereinmilling the mixed allergen composition comprises passing the mixedallergen composition through a screen with an opening size of about0.033 inches.
 5. The method of claim 1, wherein milling the mixedallergen composition comprises applying a vacuum suction through theconical mill.
 6. The method of claim 1, further comprising wetting themilled composition to a moisture content of about 6% to about 9% priorto applying microwaves or radio frequency interference to the milledmixed allergen composition.
 7. The method of claim 6, wherein the milledcomposition is wetted to a moisture content of about 9% prior toapplying microwaves or radio frequency interference to the milled mixedallergen composition.
 8. The method of claim 1, further comprisingwetting the milled composition to a water activity of about 0.65 toabout 0.8 prior to applying microwaves or radio frequency interferenceto the milled mixed allergen composition.
 9. (canceled)
 10. The methodof claim 1, wherein the milled composition is heated to at least 200° F.for at least 60 minutes.
 11. (canceled)
 12. The method of claim 1,wherein the milled composition is heated to no more than 250° F.
 13. Themethod of claim 1, wherein the milled composition is heated for no morethan 360 minutes.
 14. The method of claim 1, wherein the milledcomposition is heated in a microwave blender.
 15. (canceled)
 16. Themethod of claim 1, further comprising pressurizing the milledcomposition to at least 30 psi for at least 30 minutes.
 17. (canceled)18. The method of claim 1, wherein the substantially aerobic organismfree mixed allergen composition has at least one characteristic selectedfrom the group consisting of: fewer than about 4.5 log CFU/g totalaerobic organisms; at least about 0.5 log CFU/g fewer total aerobicorganisms than the mixed allergen composition; and fewer than about 1log CFU/g total coliforms.
 19. (canceled)
 20. (canceled)
 21. The methodof claim 1, wherein the substantially aerobic organism free mixedallergen composition has a substantially similar protein structure asthe mixed allergen composition as measured by SDS-PAGE.
 22. The methodof claim 1, wherein the substantially aerobic organism free mixedallergen composition has a substantially similar allergen effect as themixed allergen composition as measured by immune response in a patient.23. The method of claim 1, wherein the bulking agent comprises a sugar.24. The method of claim 1, wherein the mixed allergen compositioncomprises equal parts by protein mass of: at least one nut flour,wherein each nut flour is selected from the group consisting of peanutflour, almond flour, walnut flour, cashew flour, hazelnut flour, pecanflour and pistachio flour; at least one fish powder, wherein each fishpowder is selected from the group consisting of cod powder and salmonpowder; wheat; and powdered hen's egg or egg white. 25-28. (canceled)29. A substantially aerobic organism free mixed allergen compositionprepared by the method of claim
 1. 30. A food product comprising thesubstantially aerobic organism free mixed allergen composition of claim29.